Three New Species, Two New Records and Four New Collections of Tubeufiaceae from Thailand and China

Tubeufiaceae, a cosmopolitan family with a worldwide distribution, is mostly reported as saprobic on decaying woody materials from both aquatic and terrestrial habitats. The family is commonly found as helicosporous hyphomycetes, while some are chlamydosporous and phragmosporous. In this study, thirteen helicosporous hyphomycetes were collected from Thailand and China. The phylogenetic analyses of combined ITS, LSU, TEF1-α, and RPB2 sequence data placed them in Dematiohelicomyces, Helicoma, Helicotruncatum, Neohelicosporium, Parahelicomyces, and Tubeufia within Tubeufiaceae. Three new species, Tubeufia cocois, Parahelicomyces chiangmaiensis, and Neohelicosporium bambusicola, one new host record, Tubeufia laxispora, and one new geographic record, T. longihelicospora, are introduced based on both morphological characteristics and phylogenetic analyses. In addition, Dematiohelicomyces helicosporus, Helicoma guttulatum, Helicotruncatum palmigenum, and Tubeufia cylindrothecia are described with detailed descriptions and color photo plates.


Introduction
The order Tubeufiales was introduced by Boonmee et al. [1] to accommodate a single family Tubeufiaceae based on phylogenic evidence. Later, the other two families, Bezerromycetaceae and Wiesneriomycetaceae, were accepted into Tubeufiales by Liu et al. [2] based on phylogenetic analysis and divergence time estimates. Tubeufiales currently includes three families viz. Bezerromycetaceae, Tubeufiaceae, and Wiesneriomycetaceae, of which Bezerromycetaceae and Wiesneriomycetaceae are known for only sexual morphs, while Tubeufiaceae has been reported to have both sexual and asexual morphs, and the asexual morphs are mostly found as helicosporous hyphomycetes [2][3][4].

Sample Collection, Isolation, and Specimen Examination
Decaying wood, leaves, and culms were collected from Chiang Rai and Chiang Mai Provinces of Thailand from May 2020 to March 2021 and Yunnan Province of China in September 2021. Specimens were brought to a mycology laboratory for observation. Tian et al. [27,28] and Senanayake et al. [29] were followed for the morphological study and single spore isolation. Morphological characteristics were examined under a stereomicroscope (Motic SMZ-171, Wetzlar, Germany). Conidiomata were observed and photographed using a Nikon ECLIPSE Ni-U compound microscope connected with a Nikon camera series DS-Ri2. Germinating conidia were transferred aseptically to a potato dextrose agar (PDA) medium, incubated at 28 • C for 2-4 weeks, and the morphological characteristics of cultures were recorded.
Herbarium specimens were deposited at the herbarium of the Mae Fah Luang University (MFLU) and Kunming Institute of Botany (HKAS), while the living cultures were deposited at Mae Fah Luang University Culture Collection (MFLUCC) and Kunming Institute of Botany Culture Collection (KUMCC). Faces of Fungi and Index Fungorum numbers were registered as outlined in Index Fungorum [30] and Jayasiri et al. [31].

DNA Extraction, PCR Amplification, and Sequencing
Genomic DNA was extracted from two-week-old living pure cultures grown on PDA using the Biospin Fungus Genomic DNA extraction Kit (BioFlux, Kun Ming, P.R. China) following the manufacturer's protocol. DNA was subjected to PCR amplification to amplify the genes ITS, LSU TEF1-α, and RPB2, while internal transcribed spacer (ITS) with the primer pair of ITS4/ITS5 [32], the partial large subunit nuclear rDNA (LSU) with the primer pair of LR0R/LR5 [33], the translation elongation factor 1-alpha gene (TEF1-α) with the primer pair of EF1-983F/EF1-2218R [34], and RNA polymerase II second largest subunit (RPB2) with the primer pair of RPB2-5f/7cR [35]. The PCR was carried out using the method described by Tian et al. [28]. ITS, LSU, and TEF1-α amplification reactions were set using the method described by Cai et al. [36] and Lu et al. [37]. RPB2 amplification reaction was set using the method described by Lu et al. [3]. PCR products were checked and purified in 1% agarose gels and were sequenced at TsingKe Biological Technology (Kunming) Co., China.

Phylogenetic Analyses
The raw sequences (ITS, LSU, TEF1-α, and RPB2) were spliced using SeqMan and subjected to BLAST in GenBank to find closely related taxa. Sequences of four genes downloaded from NCBI GenBank are listed in Table 1. A single gene sequence alignment was generated with MAFFT v.7.110 online application [38,39] and trimmed using trimAl v 1.2 with the 'gappyout' option [40]. Multiple genes were concatenated by Sequence Matrix. Multigene phylogenetic analyses of the concatenated genes were reconstructed from maximum likelihood (ML) and Bayesian inference (BI) analyses. Maximum likelihood was performed using the online RAxML-HPC on XSEDE tool on CIPRES under the GTRGAMMA substitution model and 1000 bootstrap replicates [38,41,42]. Bayesian inference analysis was performed using the MrBayes on XSEDE tool on CIPRES [42]. The best-fit models were selected as GTR+I+G for ITS, LSU, TEF1-α, and RPB2 for the Bayesian posterior probability analysis. Two parallel runs were conducted using the default settings, six simultaneous Markov chains were run for 50,000,000 generations, and trees were sampled every 500th generation. The alignment generated in this study was submitted to TreeBASE (https://treebase.org/treebase-web/home.html, accessed on 10 January 2022) under the submission number ID29068. Trees were visualized with FigTree v1.4.4, and layouts were carried out with Adobe Illustrator CS5 v. 16.0.0.

Dematiohelicomyces helicosporus
Culture characteristics: conidia germinating on PDA within 12 h; colonies growing on PDA, reaching 20 mm in 2 weeks at 28 • C, circular, with a flat surface, edge entire, pale brown to dark brown in PDA medium; mycelium partially immersed, branched, multi-septate, hyaline to pale brown, smooth.  Figure 1). Therefore, we identify our new isolate as Dematiohelicomyces helicosporus. Chlamydotubeufia helicospora was collected on decaying wood in a flowing freshwater stream in Uttaradit Province, Thailand [43], and later, based on morphology and phylogeny, Lu et al. [3] synonymized this taxon under Dematiohelicomyces helicosporus. In this study, our new isolate was also collected from a submerged decaying wood in Chiang Rai Province, Thailand, which is a little far from the original collection location, meaning that this species still prefers similar environmental conditions.  , with H. muelleri as a type species. Two types of asexual morphs have been observed in Helicoma: the first asexual morphs are characterized by conidiogenous cells that are cylindrical, with denticles, intercalary, arising laterally from the lower portion of conidiophores, and conidia are pleurogenous, tapering Helicoma was introduced by Corda [44], with H. muelleri as a type species. Two types of asexual morphs have been observed in Helicoma: the first asexual morphs are characterized by conidiogenous cells that are cylindrical, with denticles, intercalary, arising laterally from the lower portion of conidiophores, and conidia are pleurogenous, tapering towards the apex and rounded at the tip, helicoid, hygroscopic, and become loosely coiled in water [12]. Another asexual morph is characterized by conidia that are acrogenous or acropleurogenous, helicoid, circinate, dry, tapering towards the apex, truncating at the base, coiled 1 1 /4 -3 /4 times, and not becoming loose in the water. There are 97 records listed in Index Fungorum (2021), however, most of them are lacking sequence data in GenBank. The last treatment of Helicoma was provided by Lu et al. [3], and they accepted 57 species within the genus while introducing 10 new species and 11 new combinations. In this study, the new isolate is identified as H. guttulatum based on both phylogenetic analysis and morphological characteristics. Culture characteristics: conidia germinated on PDA within 12 h; colonies growing on PDA, reaching 25 mm in 2 weeks at 28 • C, circular, with a flat surface, edge entire, and pale brown to brown in PDA medium; mycelium were partially immersed, branched, multi-septate, hyaline to pale brown, and smooth.
Notes: Helicoma guttulatum was introduced by Hyde et al. [43] on submerged decaying wood from a freshwater stream in Thailand. In our phylogenetic analyses, the newly obtained isolate (MFLUCC 21-0152) clustered with the ex-type strain of H. guttulatum (MFLUCC 16-0022) with high statistical support (100% ML/1.00 PP, Figure 1). Morphologically, the new isolate was indistinguishable from the holotype of H. guttulatum [43]. Therefore, we identify the new isolate as Helicoma guttulatum based on morphological and phylogenetic data. The monotypic genus Helicotruncatum was established by Lu et al. [3] with H. palmigenum as the type species, and it is the only species accepted in the genus [3,12]. The monotypic genus Helicotruncatum was established by Lu et al. [3] with H. palmigenum as the type species, and it is the only species accepted in the genus [3,12]. Helicotruncatum palmigenum was originally placed in Helicoma, based on morphological charac-ters [12,45,46]. Phylogenetic analysis of Lu et al. [3] showed that H. palmigenum formed an independent lineage and was distant from Helicoma. Morphologically, H. palmigenum can be distinguished from other helicosporous hyphomycetes by the distinctively thickened lateral cell wall of the conidiophore and basal cell of the conidium. Thus, Lu et al. [3] introduced a new genus, Helicotruncatum, to accommodate H. palmigenum based on both phylogeny and morphology. In this paper, Helicotruncatum palmigenum was collected from dead Cocos nucifera leaves in Thailand. Helicotruncatum palmigenum was introduced as Helicoma palmigenum by Linder [45] on decaying petioles of palms that were collected from Australia, Brazil, China, Indonesia, Japan, Mexico, New Guinea, Seychelles, Thailand, Trinidad, and the USA [3,45,46]. In addition, Helicotruncatum palmigenum has been reported on leaves and husks of Cocos nucifera [34]. Our new isolate was also collected on dead leaves of Cocos nucifera from Thailand.  Neohelicosporium was introduced by Lu et al. [10], with five new species. The taxonomic revision of the genus was recently provided by Lu et al. [3]; eight Helicosporium, two Helicoma, and one Tubeufia species were transferred to Neohelicosporium based on both phylogeny and morphology. The genus is characterized by superficial, ellipsoidal to subglobose, ostiolate ascomata, bitunicate, cylindrical, pedicellate asci and fusiform, straight or slightly curved, multi-septate, guttulate, hyaline, smooth-walled ascospores; macronematous, mononematous, branched or unbranched, septate, pale brown to brown conidiophores, holoblastic, mono-to polyblastic, integrated, sympodial, intercalary or terminal conidiogenous cells with denticles and solitary, acrogenous and/or acropleurogenous, helicoid, multi-septate, guttulate, hyaline to pale brown conidia. Species of the genus are saprobic on decaying woody substrates from both aquatic and terrestrial habitats [47]. In this study, the new species Neohelicosporium bambusicola is introduced based on both phylogenetic analysis and morphological characters.    Parahelicomyces is a well-studied genus, introduced as Pseudohelicomyces by Lu et al. [3] with Pseudohelicomyces talbotii as the type species [3,48]. Pseudohelicomyces was renamed Parahelicomyce by Hsieh et al. [49] because Parahelicomyces was a homonym and illegitimate. Currently, seven species are accepted in the genus, and all the species have sequence data available in the GenBank database. The genus is characterized by superficial, subglobose, ellipsoidal-ovate, coriaceous, ostiolate ascomata, bitunicate, cylindrical, apically thickened and rounded asci, and fusiform, multi-septate, hyaline, smooth-walled ascospores [3], as well as macronematous, mononematous, hyaline to brown, branched, septate conidiophores, holoblastic, mono-to polyblastic, integrated, intercalary or terminal, determinate or sympodial conidiogenous cells with denticles and pleurogenous or acropleurogenous, helicoid, multi-septate, hyaline to pale brown conidia. Species of the genus Parahelicomyces are found from both terrestrial and freshwater habitats in China, Japan, Mexico, South Africa, and Thailand [3,50]. In this study, we introduced a new Parahelicomyces species from Thailand.
Culture characteristics: conidia germinated on PDA within 12 h; colonies adpressed reaching 30 mm in 2 weeks at 28 • C, amd were circular, brown to dark brown, reverse brown, and slow-growing; mycelium was superficial and partially immersed, branched, septate, hyaline to pale brown, and smooth.
conidiophores [52]. Both phylogenetic analyses and morphological characteristics support Parahelicomyces chiangmaiensis as a distinct new species.  3.2.6. Tubeufia Penz. and Sacc., Malpighia 11 (11)(12): 517 (1898) Index Fungorum: IF 5635 Type species: Tubeufia javanica Penz. and Sacc., Malpighia 11 (11)(12): 517 (1898) Tubeufia, the type genus of Tubeufiaceae, was established by Penzig and Saccardo [52]. Currently, 88 records are listed in the Index Fungorum (2021); however, most of the species are lacking sequence data in the GenBank. While morphologies of Tubeufia species are quite similar, using morphology alone presents difficulties for identification; thus, sequence data are required to resolve taxonomic confusions. The last treatment of Tubeufia was provided by Lu et al. [3], and they introduced seventeen new species and six new combinations in the genus, accepting fifty species in the genus based on both phylogenic analysis and morphological characters. In this paper, we introduced two novel species, one new record species, and a new isolate of known species in Tubeufia.
Culture characteristics: conidia germinated on PDA within 12 h; colonies grow on PDA, reach 30 mm in 2 weeks at 28 • C, are irregular, with a flat surface, edge undulate, and brown to dark brown in PDA medium; mycelium are superficial and partially immersed, branched, septate, hyaline to brown, and smooth.
Material    Notes: Tubeufia cylindrothecia was originally introduced with both sexual and asexu morphs that link to Helicomyces roseus based on morphological studies [6,56], while ph logenetic analyses showed that Tubeufia cylindrotheci and Helicomyces roseus can be reco nized as two different species [1,8,54,57]. Luo et al. [54] first reported its asexual morph collected from a freshwater habitat in China. Our phylogenetic results show that t newly obtained isolate (MFLUCC 21-0160) clustered with six strains of T. cylindrothe with high bootstrap support (98% ML/1.00 PP). Morphologically, our new isolate is almo identical to T. cylindrothecia, except for the conidiogenous cells of the new isola (MFLUCC 21-0160) that are terminal or intercalary, while the conidiogenous cells are te minal in T. cylindrothecia (MFLU 16-2547). Thus, based on morphological and molecu data, we identify the new isolate as Tubeufia cylindrothecia. Notes: In our phylogenetic analyses, the newly obtained isolate (MFLUCC 21-0163) clustered with four strains of T. laxispora with high statistical supports (100% ML/1.00 PP, Figure 1). Based on pairwise nucleotide comparisons, the new strain (MFLUCC 21-0163) almost overlapped with the ex-type strain of T. laxispora (MFLUCC 16-0232), except TEF1-α 1 bp out of 878 bp (<1%). Morphologically, our new isolate fits well with the description of T. laxispora, except for the conidial size (17.5-40 µm diam., 63-94.5 µm long vs. 17.5-29 µm diam., 111-182 µm long) [37]. Hence, we identify our new isolate as T. laxispora. Tubeufia laxispora was described by Lu et al. [37] on submerged wood in Thailand, while our new isolate was collected on decaying leaves of Cocos nucifera in Thailand, and this is the first report of Tubeufia laxispora associated with a coconut tree from Thailand.
Culture characteristics: conidia germinating on PDA within 12 h; colonies growing on PDA, reaching 25 mm in 2 weeks at 28 • C, irregular, with a flat surface, edge undulate, and brown to dark brown in PDA medium; mycelium are partially immersed, branched, septate, hyaline to brown, and smooth.
Notes: Tubeufia cylindrothecia was originally introduced with both sexual and asexual morphs that link to Helicomyces roseus based on morphological studies [6,55], while phylogenetic analyses showed that Tubeufia cylindrotheci and Helicomyces roseus can be recognized as two different species [1,8,53,56]. Luo et al. [53] first reported its asexual morph as collected from a freshwater habitat in China. Our phylogenetic results show that the newly obtained isolate (MFLUCC 21-0160) clustered with six strains of T. cylindrothecia with high bootstrap support (98% ML/1.00 PP). Morphologically, our new isolate is almost identical to T. cylindrothecia, except for the conidiogenous cells of the new isolate (MFLUCC 21-0160) that are terminal or intercalary, while the conidiogenous cells are terminal in T. cylindrothecia (MFLU 16-2547). Thus, based on morphological and molecular data, we identify the new isolate as Tubeufia cylindrothecia.
Culture characteristics: conidia germinated on PDA within 12 h; colonies growing on PDA reach 20 mm in 2 weeks at 28 • C, are irregular, with a flat surface, edge undulate, and brown to dark brown in PDA medium; mycelium are superficial and partially immersed, branched, septate, hyaline to brown, and smooth. ). Therefore, we identify the three new isolates as Tubeufia longihelicospora based on morphological and phylogenetic data. Tubeufia longihelicospora was introduced by Boonmee et al. [57] on a submerged decaying wood in a small freshwater stream in Thailand. Our isolate Tubeufia longihelicospora (MFLUCC 21-0151) was also collected in Thailand, while the other two isolates of Tubeufia longihelicospora (KUMCC 21-0478 and KUMCC 21-0479) were collected in China, which is a new geographical record.

Discussion
Tubeufiaceae is an interesting family with diverse morphologies, habitats, and a worldwide distribution [1,3,12,24]. The asexual morph of the Tubeufiaceae species is reported as helicosporous, chlamydosporous, and phragmosporous conidia, of which helicosporous conidia are the most common morphology in Tubeufiaceae. However, species with helicosporous conidia are not only found in Tubeufiaceae; for example, Helicoascotaiwania also has helicosporous conidia but it is phylogenetically distinct from Tubeufiaceae. Helicoascotaiwania is placed in Pleurotheciaceae and Sordariomycetes [58], while Tubeufiaceae is placed in Pleosporales. The interesting finding is that the asexual morphs of some genera were reported with two different morphologies. For example, Tubeufia and Berkleasmium produce both dictyosporous and helicosporous conidia, while Helicoma produces both helicosporous and phragmosporous conidia, confirmed by phylogenetic analyses [3].
The morphology of helicosporous hyphomycetes is quite similar; thus, the phylogenetic analyses are efficient to identify the helicosporous hyphomycetes at the species level [59][60][61]. With the availability of molecular data, some species were revised and transferred to other genera based on phylogenetic analyses. For example, Helicomyces roseus (BCC 3381) and Helicoma perelegans (ATCC 22621) were transferred to Tubeufia [3]. However, many helicosporous species lack sequence data in the GenBank, and the taxonomy of helicosporous hyphomycetes needs revisions based on phylogenetic analyses. For example, species in the genera of Tubeufia, Helicoma, and Helicomyces were introduced based on morphological characteristics, but many of them lack sequence data in the GenBank; thus, further study into herbarium specimens is necessary to resolve taxonomic problems in the three genera.
In this study, nine Tubeufiaceae species were collected from terrestrial and freshwater habitats in Thailand, of which three were introduced as new species, while six were identified as existing species based on phylogenetic analyses and morphological characteristics. The nine species were placed in Dematiohelicomyces, Helicoma, Helicotruncatum, Neohelicosporium, Parahelicomyces, and Tubeufia, respectively, of which the genera Dematiohelicomyces, Helicotruncatum, Neohelicosporium, and Parahelicomyces are well studied, and all species in these genera have sequence data available in the GenBank. Helicoma and Tubeufia were recently revised by Lu et al. [3]. In our phylogenetic analyses, Helicoma and Tubeufia formed well-supported and monophyletics clades within the family. The morphologies of Tubeufia and Helicoma are quite similar; thus, morphology alone is not enough to identify species in Tubeufia and Helicoma, and phylogenetic analyses are necessary. However, earlier studies identified the two genera only based on morphological characteristics, and sequence data of many species are not available in the GenBank, so, it is entirely possible that some species were incorrectly identified; accordingly, fresh collections and molecular data are required to clarify their taxonomic status. Even though Dematiohelicomyces helicosporus, Helicotruncatum palmigenum, Helicoma guttulatum, Neohelicosporium bambusicola, Tubeufia cylindrothecia, T. laxispora, and T. longihelicospora are known species and were collected again, some species are known as new hosts and new geographical records. In addition, it is also better to provide the full descriptions and color plates of the micro-characteristics of new isolates to understand some fine morphological differences.